Personalized, preventative, predictive medicine demands diagnostic tests that can be performed at the point of care of an individual, with extreme fidelity that allows a caregiver or researcher to identify specific biomolecules even if those biomolecules are in low concentrations. While multiplexed measurement platforms, such as protein arrays, are utilized in research, there is a need in the art for a point of care platform that operates not only simply, but with great fidelity, as the concentration of different biomolecules in the blood can vary by more than ten orders of magnitude. As such, the identification of low concentration biomolecules, such as biomarkers of interest in certain diagnostic tests, requires that the affinity of the capture molecule to the low concentration biomolecule be orders of magnitude higher than any other molecule in the sample.
It will be appreciated that, depending upon the particular diagnostic test or assay, the substance tested may be human body fluids such as blood, serum, saliva, biological cells, urine, or other biomolecules. Additionally, testing may be further desired on consumables such as milk, plant tissues or extracts, baby food, or even water. As such there is a need in the art to provide a low cost multiplexed assay for target molecules with sufficient sensitivity and specificity.
Current attempts to provide such an assay or diagnostic test utilize affinity based sensors. Affinity based sensors function according to a “key-lock” principal in which a molecule with very high association factor to the marker of interest is used for detection. For example, a pregnancy test kit may incorporate a monoclonal antibody specific to a β-subunit of hCG (βhCG). The antibody is conjugated with a tag, e.g., gold, latex, or fluorophore, which is used for detection. If the targeted molecule binds with the conjugated antibody, the tagged pair is detectable such as by a visible test line.
Similarly, molecular tests based upon enzyme-linked immunosorbent assay (“ELISA”) techniques utilize an antibody or antigen bound to a substrate to immobilize a target molecule. For example, FIG. 1 depicts an ELISA assay 10 wherein antibodies 12 are immobilized on a substrate 14. The substrate 14 may be positioned within a well (not shown). A blocker 16 is provided to cover the surface of the substrate around the antibody 12. In a typical ELISA assay, a sample is then added to the well in which the primary antibody 12 is immobilized. Next, the sample is incubated for some time. During incubation, the blocker 16 prevents the target molecules in the sample from binding to the surface of the substrate 14 in order to avoid false binding. During incubation, some of the target molecules 18 become bound with some of the antibodies 12 as depicted in FIG. 2. After incubation, the remaining sample is washed to remove the unbound primary antibodies 18.
Subsequently, a secondary antibody 20 with a bound label 22 is added to the well, incubated, and washed resulting in the configuration of FIG. 3. As depicted in FIG. 3, the labeled secondary antibodies 20 are bound to the target molecules 18 that are in turn bound to the antibodies 12. Accordingly, the number of labels 22 bound by the antibodies 20 to the antigen 18 is proportional to the concentration of the target antigen. Depending on the label used, the number of labels can be finally detected using colorimetry, amperometry, magnetometry, voltammetry, luminescence, or fluorescence detection. Other label-free antibody processes such as surface plasmon resonance may alternatively be used.
It will be appreciated that the reliability and minimum detectable concentration of a target molecule are directly related to the sensitivity and cross-reactivity of the detection assay. Indeed, as the cross-reactivity of the assay increases, the minimum detectable concentration and the diagnosis error rate increase. The sensitivity in such tests is generally limited by label detection accuracy, association factor of the antibody-antigen pair, and the effective density of the probe antibody on the surface.
As noted above, one issue that arises with affinity based sensors is the cross-reactivity of the sensor to other biomolecules. Indeed, in cross-reactive assays, a sensor tends to also sense biomarkers other than the biomarker of interest. The cross-reactivity issue is depicted in FIG. 4 wherein an ELISA assay 30 includes antibodies 32 immobilized on a substrate 34 to act as a capture molecule with a blocker 36 covering most of the substrate surface 34. Additionally, a labeled secondary antibody 38 is bound to a target molecule 40 which is in turn bound by the primary antibody 32. The labeled secondary antibody 38 has also bound to a molecule 42 which exhibited an affinity for the primary antibody 32 and was labeled by a secondary antibody 38. The sensitivity to a broad range of biomarkers thus increases the false negative/positive rate of diagnostic tests at clinical level as reported, for example, by P. A Benn et al., “Estimates for the sensitivity and false-positive rates for second trimester serum screening for Down syndrome and trisomy 18 with adjustment for cross identification and double positive results,” Prenatal Diagnosis, Vol. 21, No. 1, pp 46-51, 2001. The presence of other molecules (secondary molecules or antigens) in the sample thus affects the minimum detectable concentration by binding to the primary antibody.
The accuracy of the assay may further be affected by physiosorption. As further depicted in FIG. 4, some features 44 present in the ELISA assay 30, either contaminants or simply an incongruity, may also be bound to a labeled secondary antibody 38. The physiosorbed labeled secondary antibody 38 thus causes an increased background signal.
Provision of diagnostic tests with a high fidelity is further complicated by the relative scarcity of the target molecules in a particular sample. As reported by Robert F. Service, “PROTEOMICS: Proteomics Ponders Prime Time,” Science 321, no. 5897 (Sep. 26, 2008): 1758-1761, the concentrations of different proteins in blood varies by more than 10 orders of magnitude. Thus, to ensure a desired level of fidelity, the affinity of the capture molecule to the biomarker of interest must be orders of magnitude higher than the affinity of the capture molecule to any other molecule in the sample.
Overcoming the cross-reactivity and background problems can significantly delay development of a new assay test and can increase the cost and complexity of the overall test. For example, in an effort to mitigate the various sensitivity and interference issues involved with affinity based testing, a particular assay is typically optimized by finding a combination of reagents and environmental conditions that maximizes the binding of the target molecule to the antibody. Thus, optimization can entail incorporating highly selective antibodies. Accordingly, a typical development of an ELISA assay requires several scientists working for more than a year to identify an acceptable antibody. Cross-reactivity of proteins is a common source of the failure of such development efforts.
Another approach for optimizing the diagnostic test for a particular target molecule entails controlling the test conditions locally at different sites of the platform to increase the specificity of the tests. One such approach is described in U.S. patent application Ser. No. 12/580,113, filed on Oct. 15, 2009, the entire contents of which are herein incorporated by reference. Control of the test conditions locally at different sites of the platform can also be used to increase the dynamic range of the assay as described in U.S. patent application Ser. No. 12/688,193, filed on Jan. 15, 2010, the entire contents of which are herein incorporated by reference.
Control of the test conditions locally at different sites of the platform has generally been attempted by electrically influencing biochemical reactions. Various attempts at such electrical control have been reported by R. G. Sosnowski et al., “Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control,” Proceedings of the National Academy of Sciences of the United States of America 94, no. 4 (Feb. 18, 1997), Ian Y. Wong and Nicholas A. Melosh, “Directed Hybridization and Melting of DNA Linkers using Counterion-Screened Electric Fields,” Nano Letters 0, no. 0 (January), Ian Y Wong, et al., “Electronically Activated Actin Protein Polymerization and Alignment,” Journal of the American Chemical Society 130, no. 25 (Jun. 1, 2008): 7908-7915, and Ulrich Rant et al., “Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets,” Proceedings of the National Academy of Sciences 104, no. 44 (Oct. 30, 2007): 17364-17369, among others.
Controlling test conditions by electrically influencing biochemical reactions, while promising, has proven problematic to researchers. For example, while influencing test conditions locally at different sites of the platform can increase the specificity of the tests or increase the dynamic range of the assay, most of the electrical potential is dissipated at the electric double layer formed over the electrode surface. Therefore, the electrical influence has a limited range of effectiveness.
A need exists for a device and method of performing an assay incorporating low cost antibodies. A further need exists for tests such as multiplexed assays, e.g., protein arrays, competitive assays, or bead based arrays, as well as low cost devices, e.g., lateral flow devices, or other biochips. Furthermore, methods and compositions for implementing these assays and arrays with higher fidelity would be appreciated. Additionally, methods and devices which provide more accurate results than previous assays would be a further benefit.